Polymerase Chain Reaction (PCR) is a powerful and widely used technology for amplifying nucleic acid sequences. PCR uses a polymerase such as Thermus aquaticus (Taq) polymerase to replicate or amplify copies of a particular segment of a nucleic acid. The number of copies of the nucleic acid increases exponentially, at least initially, and then tapers off as reactants become limiting or otherwise interfere with replication.
Conventional PCR assays are used to amplify a nucleic acid in a sample for use in, for example, analyses such as genotyping that require relatively large amounts of material. Conventional PCR assays can also be used in clinical applications to detect the presence in a cell or tissue sample of a particular pathogen or protein—for example, to detect unique mRNA transcripts from abnormal cells in a background of normal cells.
For many diseases, a quantitative measurement is needed to make a proper diagnosis, such as when a pathogen is normally present, a gene is normally expressed at low levels, or a virus is maintained endogenously in a healthy cell or tissue. Precise quantitative measurements are needed, for example, to diagnose certain infectious diseases, cancers, and autoimmune diseases, and may be useful therapeutically to assess the response of a disease to treatment and make prognoses for recovery. Precise quantitative measurement may also help detect false positives, which can occur if there is any contamination of a sample.
It can be difficult, however, to quantitate the results of any particular PCR because data from separate experiments or amplifications are typically not comparable. Amplification is an exponential process, so small differences in any of the variables which affect the reaction rate—including the length and sequence of the primer pairs, the concentration of reactants such as template and nucleosides, and the conditions for the reaction—can lead to dramatic differences in the amplification and ultimate yield of PCR product. In short, every PCR will have different reaction dynamics.
Two general approaches have made it possible to control for much of the variability among PCRs and get quantitative or at least semi-quantitative data for nucleic acids: (1) co-amplification or “competitive” PCR and (2) modeling of PCR amplification or “growth curves.”
Co-amplification is the amplification in the same mixture of two nucleic acids, such as a target nucleic acid (“target”) and a standard nucleic acid (“standard”). By conducting the PCRs of the target and standard simultaneously and under the same conditions, the effect of variables such as those noted above can be controlled, since the target and standard undergo the same treatment and experience the same conditions. Ideally, the same primers are used to amplify the target and the standard, thereby controlling for differences in efficiency due to differences in the annealing of the primers.
The relative amount of two nucleic acids can be determined by using, for example, heterogeneous methods such as agarose gel electrophoresis, Northern blot analysis, or differential display. In a co-amplification assay, a known concentration, and hence a known quantity or amount, of standard can be added to a sample containing an unknown amount of target to form a mixture and the two nucleic acids are amplified. The quantity of target can then be approximated by comparing the amount of target and standard for various concentrations of sample. The concentration of standard for which there are similar amounts of target and standard product after amplification provides an estimate of the initial amount of target.
The development of various probes has made it possible to analyze a PCR product with homogeneous methods, in real-time—that is, as the reaction proceeds. For example, some existing technologies use sequence-specific oligonucleotides to detect a product at each PCR cycle by measuring fluorescence emission. The measurements of fluorescence correspond to the number of copies of the nucleic acid. For example, TaqMan® probes utilize energy transfer fluorescence methods, in which the probes are self-quenched until cleaved during the PCR nuclease assay. With real-time sampling of a PCR, a growth curve can be fit to measures of the relative amounts of a nucleic acid at various cycles. A calibration curve, created from amplification of known quantities of the nucleic acid, can then be used to quantify the initial amount of the nucleic acid. See U.S. Pat. No. 6,503,720, “Method for quantification of an analyte.”
Different combinations of fluorescent dyes can be used to simultaneously monitor in real-time the co-amplification of nucleic acids, and the absolute amount of target can then be determined by creating a standard curve for the standard nucleic acid. The standard curve can be generated, for example, by plotting the amount of the standard produced by some cycle in a PCR against varying, but known, amounts present before amplification. See U.S. Pat. No. 5,476,774, “Quantitation of nucleic acids using the polymerase chain reaction.”
Although co-amplification of nucleic acids allows direct comparison and monitoring of different products during PCR, thereby providing data for use in quantitation, it can introduce certain artifacts into the data. For example, competitive effects may mean that linear calibrations are not accurate. Even in non-competitive methods, a change in the annealing temperature during PCR can cause a change in fluorescence signal and hence a shift in the baseline, and random effects, such as bubbles in a sample, can cause spikes in output data. Failure to identify and remove or correct these artifacts and anomalous features of the data can lead to misinterpretations of the PCR. In particular, failure to correct anomalies can result in false positives, and failure to account for the artifacts of a co-amplification will significantly reduce the precision and accuracy of quantitation based on co-amplification PCR data.